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Abstract

Three-dimensional fluorescence lifetime microscopy is achieved by combining wide-field fluorescence lifetime imaging with a remote optical refocusing method. As required for some applications in dynamic research for physics, chemistry, or biology, it is thereby not necessary to move the sample, i.e., the specimen is not disturbed during measurement. Using a fluorescent microsphere the performance of the system has been tested successfully with respect to three-dimensional fluorescence lifetime microscopy as well as time-resolved fluorescence spectroscopy.

(i) 3D fluorescence intensity image sections of a fluorescent microsphere as test sample with the mirror M on different positions along the optical axis. The gating time of the ICCD is 200 ps. (ii) 3D image of the sample reconstructed from these sections; (iii) 3D lifetime image of the sample reconstructed from 2D lifetime image sections with the mirror M on different positions.

(a) 2D fluorescence lifetime images of section (e) in Fig. 2 (i). (b) Decay curve of the fluorescence intensity; squares are experimental data points, solid curve is a single exponential fit to the data. The decay time is 4.1 ns.